Nasal powder formulation for treatment of hypoglycemia

ABSTRACT

The present invention provides a powder formulation containing glucagon or a glucagon analog for nasal administration, useful in the treatment of hypoglycemia, and in particular the treatment of severe hypoglycemia. The present invention also provides a method of making this powder formulation, and to devices and methods for using the powder formulation.

FIELD OF THE INVENTION

This application relates to a powder formulation containing glucagon ora glucagon analog for nasal administration, useful in the treatment ofhypoglycemia, and in particular the treatment of severe hypoglycemia.The application further relates to a method of making this powderformulation, and to devices and methods for using the powderformulation.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is submitted as anxml file of the sequence listing named “083389_01978.xml” which is 2,001bytes in size and was created on May 3, 2023. The sequence listing iselectronically submitted via Patent Center and is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Diabetes has reached epidemic proportions in much of the western worldand is a serious and growing public health concern in many developingeconomies. Globally, there are approximately 285 million people withdiabetes and that number is expected to reach 438 million by 2030 (IDFDiabetes Atlas, 2009.)

Diabetes complications are usually associated with chronically elevatedblood glucose levels (hyperglycemia), which result in heart, kidney andeye diseases, amputations and neurological impairment. Unfortunately,there are very real and serious complications associated with use ofmedications used to treat the diabetes-related hyperglycemia. One of themost common complications of treatments used to reduce blood sugarlevels is hypoglycemia (low blood sugar), most frequently seen inpatients being treated with insulin (i.e., all persons with type 1diabetes and approximately 30% of patients with type 2 diabetes) butalso in patients with type 2 diabetes receiving sulfonylurea treatment.Indeed, if it was not for the barrier of hypoglycemia, people withdiabetes could probably have normal blood glucose levels and thus avoidthe complications associated with hyperglycemia (Cryer, 2002).

Depending on the severity of the episode, hypoglycemia causes a widerange of physical problems ranging from weakness, dizziness, sweating,chills and hunger to more serious symptoms including blurred vision,behavior change, seizures, coma and even death. In addition to thephysical effects of hypoglycemia, there are significant psychologicaleffects including embarrassment, fear of another episode, high levels ofanxiety and low levels of overall happiness that adversely affectglucose control and quality of life (Deary, 2008).

Severe hypoglycemia in a conscious person should be treated by the oralingestion of carbohydrate, preferably as glucose tablets or equivalent.For severe hypoglycemia in an unconscious individual outside of thehospital setting, the recommended treatment is 1 mg of glucagon byintramuscular (IM) or subcutaneous (SC) injection. For severehypoglycemia in an unconscious individual in the presence ofprofessional medical assistance and intravenous access, intravenousdextrose is recommended. In all cases, once the hypoglycemia has beenreversed, the patient should be given access to oral carbohydrates tofully recover and prevent repeated hypoglycemia.

Glucagon, a highly effective treatment for severe hypoglycemia bothoutside and within the hospital setting, is currently available only asa powder that must be mixed with a diluent immediately prior toadministration by injection. Although this is a procedure that would berelatively easy for people with diabetes who inject insulin, they arenot treating themselves because, by definition, severe hypoglycemia is ahypoglycemic episode in which the patient requires third partyassistance (Cryer, 2009). For any non-medical person who is confrontedwith an emergency situation in which a patient with diabetes is in ahypoglycemic coma or suffering hypoglycemia-related convulsions,reconstitution and injection of the current injectable glucagon is acomplex and daunting procedure that is fraught with potential forerrors.

Indeed, Australian researchers have published a study in which parentsof children and adolescents with diabetes used one of the currentlyavailable glucagon kits (GlucoGen Hypokit, Novo Nordisk) in a simulatedemergency situation (Harris et al, 2001). Each parent was asked topretend it was 3:00 am and their child was unconscious. They were thengiven an unopened emergency glucagon kit and asked to administer themedication in a wrapped piece of meat to simulate a thigh. A small ofgroup of 11 diabetes health professionals (five endocrinologists and sixdiabetes educators) served as surrogate control.

Of the 136 parents who participated in the study, 106 were parents ofteenagers with a mean duration of diabetes of 4.7 years and 30 wereparents of younger children with a mean duration of diabetes of 2.4years. Over 90% reported having been previously trained on use ofglucagon. Fully 69% of these parents experienced difficulties handlingthe current glucagon emergency kit. Difficulties included difficulty inopening the pack, removal of the needle sheath, mixing of theingredients and bending of needles. On average, these parents required 2minutes and 30 seconds to complete the procedure (range 30 seconds to >12 minutes). In addition, 6% aborted the injection entirely and 4% ofthe participants injected only air or only diluent. In contrast,diabetes professionals performed the procedure in 1 minute and 17seconds (range 1-1.75 minutes). The number of errors observed in thissample of parents is disconcerting especially in light of the fact thatthis was a timed simulation and not a true emergency.

Difficulties associated with use of the glucagon emergency kit arecorroborated in a recent report from the Institute for Safe MedicationPractices (ISMP) Canada (ISMP Canada Safety Bulletin, 2010). The ISMPreport of September 2010 documents three separate incidents in which thediluent was administered on its own, without the glucagon powder havingbeen reconstituted with the diluent before administration. This resultedin complete failure to deliver the intended dose of glucagon toindividuals experiencing a severe hypoglycemic crisis and, according tothe report, resulted in patient harm in one of the cases.

A telephone survey was conducted with 102 patients with type 1 diabetesto ascertain their opinions on the currently available glucagonemergency kits (Yanai, 1997). Most patients (67%) stated they wouldprefer an intranasally administered glucagon were it available and fully82% of these patients assumed family members, teachers and colleagueswould prefer to administer emergency therapy by the intranasal route.Likewise, amongst emergency care professionals who are frequently thefirst to be called to treat a patient suffering from an episode ofsevere hypoglycemia, there is significant concern regarding the injectedroute of administration. Inherent in using sharps, there is the veryreal risk of accidental blood exposure and needlestick and theassociated potential for contracting life-threatening infectiousdiseases (Leiss J 2006). Within this context, some emergencyprofessionals are actively seeking noninvasive routes of administration,including intranasal, as a means to enhance emergency patient care,increase patient and care-giver safety while increasing the pool of careproviders who can effectively respond to the emergency (Curran, 2007).

These considerations make it clear that the present approach to theadministration of glucagon in emergency situations is lacking, and thatthere exists a real need for alternative approaches for deliveringglucagon to treat severe hypoglycemia.

Various approaches to delivery of glucagon via intranasal administrationhave been proposed but they have not resulted in the availability of anapproved alternative to injected glucagon. In general, these approachescan be divided into two groups, those that use administer a liquidformulation, and those that use some type of dry formulation.

Within the liquid formulations group, the compositions used in Pontiroli(1983), Pontiroli (1985), Freychet (1988), Pontiroli (1989), Pontiroli(1993) and Pacchioni (1995) were all formulations that needed to besprayed into the nose. More recently, Sibley et al., 2013, reportedsuccessful use of what was intended to be injectable glucagon byspraying the reconstituted glucagon solution intranasally in a patientin the out-of-hospital environment.

Because glucagon is not stable in the liquid state, the liquidcompositions used in these studies needed to be reconstitutedimmediately prior to use and are therefore not ideal for emergency usein treating severe hypoglycemia. Further, in many of these studies,patients needed to take a deep breath immediately after dosing withthese compositions. Since patients with severe hypoglycemia arefrequently unconscious or even comatose, they cannot be asked to take adeep breath. As such, these compositions are not ideal for intranasaldelivery for treatment of severe hypoglycemia, and do not overcome thechallenges of injectable formulations that involve use of a needle bynon-medical professionals and need to be prepared prior to use.

Within the second group, U.S. Pat. No. 5,059,587 discloses powders fornasal administration of physiologically active peptides, includingglucagon. These powders include a water-soluble organic acid as anabsorption promoter.

Jorgensen et al. 1991 disclosed a “powdery formulation of glucagon fornasal delivery.” This formulation is disclosed as containing glucagon,didecyl phosphatidylcholine (DDPC) and α-cyclodextrin (α-CD), and isreported as providing a dosage dependent response with respect toincreases in plasma glucose and plasma glucagon. No compositionalamounts or method of making the formulation are disclosed in thisreference.

The Jorgensen 1991 formulation or HypoGon ® Nasal (NovoNordisk) isidentified as the material used in several subsequent studies, and inone of these reports the formulation is said to have a composition ofglucagon:DDPC: α-CD in a 5:10:85 ratio by weight. In these studies,intranasal administration to adults of the Jorgensen 1991 powderformulation is reported to show an increase in plasma glucoseconcentration in adults with hypoglycemia. In these studies, glucoselevels increased after dosing to reach a plateau at about 30 minutesafter dosing. In contrast, treatment with injected glucagon in thesestudies resulted in glucose levels that continued to increase from thetime of administration for up to at least 90 minutes (Hvidberg, 1994;Rosenfalck, 1992). Intranasal administration to children withhypoglycemia of the Jorgensen 1991 powder formulation is reported toincrease plasma glucose concentration soon after dosing to peak levels25-30 minutes post-dosing after which glucose levels decreased. Incontrast, treatment of children with injected glucagon resulted inplasma glucose levels that continued to rise for at least 45 minutes(Stenninger, 1993).

Sakr, 1996 reports a comparison of spray and powder formulationscontaining glucagon and dimethyl-β-cyclodextrin (DMβCD). Nasal spray wasprepared by dissolving commercial glucagon in the “manufacturer’ssolvent” containing 2 or 5% w/v DMβCD. Powders were obtained by freezedrying of the spray solutions.

Teshima et al (2002) found that a maximum plasma glucose increase of1.56 mmol/L (28.08 mg/dL) in healthy volunteers upon intranasaladministration of a powder containing glucagon and microcrystallinecellulose at a ratio of 1:69. They also reported that the powder form isstable at 5 and 25° C. for at least 84 days. For an intranasal productin patients with insulin-induced hypoglycemia, an increase of only 1.5mmol/L may be inadequate to bring the patient back to normal bloodglucose levels. In addition, the volume of powder (i.e., 70 mg for a1:69 ratio formulation) is considerable and may be excessive for usewith available devices.

Matilainen et al (2008, 2009) investigated the solid-state stability anddissolution of glucagon/ γ-CD and glucagon/lactose powders at anincreased temperature and/or humidity for up to 39 weeks, with the solidstate stability of the glucagon/ γ-CD powder being better. The powderwas not used for intranasal administration.

Endo et al (2005) reported that the use of erythritol as both anexcipient and a carrier in a dry-powder inhaler of glucagon forpulmonary administration. The powder was formulated by mixing micronizedglucagon particles and excipients with larger carrier particles. Toachieve alveolar deposition for subsequent systemic absorption, a drypowder inhalant (DPI) of glucagon was size-reduced to a mass mediandiameter between 1 and 6 micron, as measured by laser diffractionanalysis.

Onoue et al (2009) reported that addition of citric acid in glucagondry-powder inhaler for pulmonary inhalation improved the dissolutionbehavior, and did not impair the solid-state stability. Intratrachealadministration of glucagon dry-powder inhaler (50 µg/kg in rats)containing citric acid led to 2.9-fold more potent hyperglycemic effectin rats, as compared to inhaled glucagon without citric acid. Both theEndo (2005) and Onoue (2009) disclosures present pulmonary delivery ofglucagon. As patients with severe hypoglycemia may be unconscious orseverely disoriented, they cannot be expected to breathe deeply toassure pulmonary delivery. As such, pulmonary delivery of glucagon isnot appropriate for treatment of severe hypoglycemia.

Notwithstanding these efforts, no current product is available topatients that utilizes a nasal powder to administer glucagon for thetreatment of severe hypoglycemia.

It is an object of the present invention to provide such a nasal powderformulation.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, a powderformulation of glucagon or a glucagon analog is provided. This powdercomposition comprises glucagon or a glucagon analog, a cyclodextrin, anda phospholipid surfactant, and is formulated such that at least aportion of the powder is present in a phase characterized by an XRPDmesopeak as determined by x-ray powder diffraction. In further specificembodiments, the powder composition consists of:

-   (a) 5 to 15 wt% glucagon or glucagon analog;-   (b) 5 to 51 wt% of phospholipid surfactant;-   (c) 44 to 90 wt% of cyclodextrin and-   (d) optionally, up to 10 wt% of a low molecular weight organic acid,    or a pharmaceutically acceptable water soluble salt of ester    thereof.

In accordance with a second aspect of the invention, a nasal applicatorfor a powder formulation is provided. The applicator includes a powderformulation reservoir, and a powder formulation in accordance with theinvention contained within the reservoir

In accordance with a third aspect of the invention, a method for makingthe powder formulation of the invention is provided. This methodcomprises the steps of :

-   (a) forming a first mixture of the glucagon and the surfactant in an    aqueous carrier, wherein the surfactant is present at a    concentration greater than or equal to the critical micelle    concentration;-   (b) adding the cyclodextrin to the first mixture to form a second    mixture;-   (c) drying the second mixture to form a solid formulation; and-   (d) processing the solid formulation to produce a uniform powder,    said uniform powder including at least a portion of the powder in a    phase characterized by an XRPD mesopeak. In specific embodiments,    the drying of the second mixture may be carried out by freeze drying    or spray drying the second mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Plasma glucose concentration in mmol/L over time uponintranasal administration to dogs at a 1 mg dose of glucagon via apowder formulation with a glucagon:DPC: β-CD weight ratio of 10:10:80.

FIG. 2 : Plasma glucose concentration in mmol/L over time uponintranasal administration to dogs at a 1 mg dose of glucagon via apowder formulation with a glucagon:DDPC: β-CD weight ratio of 10:10:80.

FIG. 3 : Plasma glucose concentration in mmol/L over time uponintranasal administration to dogs at a 1 mg dose of glucagon via apowder formulation with a glucagon:LLPC: β-CD weight ratio of 10:10:80.

FIG. 4 : Plasma glucose concentration in mmol/L over time uponintranasal administration to dogs at a 750 µg dose of glucagon via apowder formulation with a glucagon:D8PC: β-CD weight ratio of 10:10:80.

FIG. 5 : Plasma glucose concentration in mmol/L over time uponintranasal administration to a single nostril of dogs at a 750 µg doseof glucagon via a powder formulation with a glucagon:DLPG: α-CD weightratio of 5:25:70.

FIG. 6 : X-ray powder diffractograms of powder formulations of glucagon:DPC: β-CD and of glucagon-DDPC- β-CD at a weight ratio of 10:10:80.

FIG. 7 : Average plasma glucose concentrations in adults with type 1diabetes and insulin-induced hypoglycemia treated with intranasal andinjected glucagon.

FIG. 8A: Average plasma glucagon concentrations in children, ages 12-17,with type 1 diabetes treated with intranasal and injected glucagon. Topline is intramuscular, bottom line is intranasal.

FIG. 8B: Average plasma glucose concentrations in children, ages 12-17,with type 1 diabetes treated with intranasal and injected glucagon. Topline is intramuscular, bottom line is intranasal.

FIG. 9A: Average plasma glucagon concentrations in adults with (topline) and without nasal congestion (bottom line), and with congestionand pre-treatment with a nasal decongestant (middle line).

FIG. 9B: Average plasma glucose concentrations in adults with (top line)and without (bottom line) nasal congestion, with congestion andpre-treatment with a nasal decongestant (middle line).

FIG. 10 : Exemplary application device for nasal powder formulations(Aptar device).

DETAILED DESCRIPTION OF THE INVENTION

Some of the desired attributes for an intranasal powder formulation withcommercial potential are listed below.

-   Uniform dose deliverability by a device for intranasal    administration-   Absence of a significant fraction of small particles to preclude    inadvertent administration to the lungs-   Adequate drug content to provide the total dose of drug required to    achieve therapeutic effect as a single dose into a single nostril-   Adequate drug content to deliver the total dose in a few tens of    milligrams, or the maximum allowed by the delivery device-   Adequate drug content and absorption characteristics to be effective    despite the presence of nasal congestion that may be associated with    allergies or common cold-   Stability during storage under ambient conditions for an extended    period of time, preferably at least 18 months-   Good safety and tolerability profile

Previous attempts at developing an intranasal powder formulation fallshort in one or several of the desired attributes.

Compositions described in this invention are designed to meet some andpreferably all of these desired attributes in compositions having threerequired components: glucagon or a glucagon analog, a cyclodextrin, anda phospholipid surfactant .

GLUCAGON AND GLUCAGON ANALOGS

As used in the specification and claims of this application, “glucagon”refers to a polypeptide of the sequence

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr (SEQ ID NO: 1).

The glucagon may be chemically synthesized, produced by recombinant DNAtechnology or extracted from natural sources. The term “glucagon analog”refers to variants of this sequence that retain the ability to stimulateincrease in blood glucose in vivo but which may offer benefits forpharmaceutical uses such as greater activity, greater solubility orgreater stability.

Examples of glucagon analogs in which one amino acid of the naturalsequence is replaced with an alanine as well as analogs with multiplesubstitutions are disclosed in Chabenne et al., (2014), which isincorporated herein by reference. An exemplary analog in which threeamino acids are modified to result in a glucagon analog with enhancedbiological activity is [Lys^(17,18), Glu²¹] glucagon. Zealand Pharma hasdisclosed a multitude of glucagon analogs for example in U.S. Pat.Publications 20140080757, 2014001733, 20130316941, 20130157935,20130157929, 20120178670, 20110293586, 20110286982, 20110286981, and20100204105 which are incorporated herein by reference. These analogsare reported to have greater binding affinity for the GLP receptor thanthe glucagon receptor, but nonetheless retain the activity of glucagon.Zealand Pharma has also commenced clinical trials of a glucagon analogfor treatment of hypoglycemia designated as ZP4207. U.S. Pat.Publication 20130053310, which is incorporated herein by reference,discloses other glucagon analogs useful in treatment of hypoglycemia.

Phospholipid Surfactants

Phospholipids are ubiquitous components of biological membranes that arepart of cells and tissues in the human body, including the nasal mucosa.The most prevalent phospholipid surfactants in cell arephosphatidylcholines and phosphocholines (PC), althoughphosphatidylglycerols (PG) are significant components of biologicalmembranes.

PCs and PGs may be used in the formulations of the invention.Lysophosphospholipids derived from a diacyl PC or PG by removal one ofthe acyl groups may also be used. Preferred phospholipids are soluble inwater or acidified water, although a pharmaceutically acceptablecosolvent such as ethanol, dimethylsullfoxide or N-methylpyrrolidonecould be used if needed to enhance phospholipid solubility.

In accordance with the present invention, exemplary phospolipidsurfactants that may be employed in the powder formulation aredodecylphosphocholine (DPC), 1,2-didecyl-sn-glycero-3-phosphocholine(DDPC or “didecylphosphatidylcholine”),1-didecanoyl-sn-glycero-3-phosphocholine (LLPC or“lysolauroylphosphatidylcholine”),1,2-dioctanoyl-sn-glycero-3-phosphocholine (D8PC or“dioctanoylphosphatidylcholine”) and1,2-dilauroyl-sn-glycero-3-phospho(1′-rac-glycerol) (DLPG or“dilauroylphosphatidylglycerol”).

Preferred phospholipid surfactants are those that form micelles, ratherthan bilayers at the concentration used during manufacture of the powderformulation. This includes DPC, DDPC, LLPC, and D8PC, but not DLPG.

In general, the formation of micelles as opposed to bilayers can bepredicted based on the structure of the phospholipid surfactants, whichare made of two or three parts: a phosphorous-containing choline orglycerol headgroup, an optional glycerol backbone and one or two acylchains. The length of the acyl chain and number of acyl chains permolecule are important in determining whether a certain phospholipidforms a micelle or a bilayer. Where only one acyl chain is present, asin DPC and LLPC which have 12 carbons in their single acyl chain,micelles are likely to be formed as opposed to bilayers provided thelength of the acyl chain is less than 14. Diacyl phospholipids containtwo acyl chains per molecule. When the chain length of each chain isless than 12, they tend to form micelles. DLPG, DDPC and D8PC are diacylphospholipids. DLPG contains 12 carbons per acyl chain and formsbilayers. DDPC contains 10 carbons per acyl chain. It forms eitherbilayers or micelles depending on the concentration (Marsh, 1990). D8PCcontains 8 carbons per acyl chain, and mostly forms micelles.

In specific embodiments of the invention, the formulation contains asingle type of phospholipid surfactant. In other embodiments, thephospholipid surfactant component of the formulation can be made up frommixtures of phospholipid surfactants, including for example, acombination of any two, three or four of the surfactants identifiedabove.

Cyclodextrins

Cyclodextrins as a class are composed of 5 or more α-D-glucopyranosideunits linked 1->4, as in amylose (starch). As used in this application,however, the term “cyclodextrins” refers to the more common and typicalcyclodextrins containing six, seven or eight glucose residues in thering creating a cone shape, namely:

-   α (alpha)-cyclodextrin: 6-membered sugar ring molecule-   β (beta)-cyclodextrin: 7-membered sugar ring molecule-   γ (gamma)-cyclodextrin: 8-membered sugar ring molecule

α-CD was used in the powder formulation (HypoGon® Nasal) by Novo Nordiskin clinical trials (Stenniger and Aman, 1993; Rosenfalck, 1992). Theaqueous solubility of α-CD is reported to be about 5 wt%.

Two other cyclodextrins, one with aqueous solubility less than that ofα-CD (ß-CD, 1.85 wt%) and another with a higher aqueous solubility thanα-CD (HP-ß-CD) are also suitable for use in the compositions of theinvention, as is γ (gamma)-cyclodextrin which is freely soluble inwater.

Cyclodextrins in the compositions of the invention act as a filler, andalso adhere to the nasal mucosal surface and aid in the absorption ofglucagon. Upon delivery to the nostril, the major ingredient (90% to 70%by weight) namely, the cyclodextrin helps the powder adhere to themucosal surface. The less soluble the cyclodextrin is, the longer thepowder is expected to be physically muco-adhesive. Based on thesolubility properties of cyclodextrins, the muco-adhesiveness isexpected to decrease in the order ß-CD > α-CD > HP-ß-CD. Because ofthis, the most preferred filler is β-CD.

The cyclodextrins of the invention may be used individually, or asmixtures of any two or more cyclodextrins.

Powder Formulation

The powder formulation of the invention contains the three ingredients,the glucagon or glucagon analog, the phospholipid surfactant and thecyclodextrin in amounts that are effective to provide a therapeuticamount of glucagon or glucagon analog in an amount of powder that can beadministered in a single dose in a single nostril. In specificembodiments, the powder formulation consists of:

-   (a) 5 to 15 wt% glucagon or a glucagon analog;-   (b) 5 to 51 wt% of phospholipid surfactant;-   (c) 44 to 90 wt% of cyclodextrin and-   (d) optionally, up to 10 wt% of a low molecular weight organic acid,    or a pharmaceutically acceptable water soluble salt of ester    thereof.

As reflected in the examples below, powder formulations of this typehave excellent properties when used to administer glucagon intranasally,yet a similar set of ingredients in the Jorgensen 1991/Rosenfalck1992/HypoGon® Nasal product did not provide comparable results and wasapparently abandoned following initial testing in human subjects.

Based on the various descriptions in the art, it is believed theJorgensen 1991/Rosenfalck 1992/HypoGon® Nasal product containedglucagon, DDPC and alpha-CD in a weight ratio of 5:10:85. No informationis provided about how these ingredients were combined. Thus, a directcomparison of this formulation with the formulation of the invention isnot possible. However, the available data for each formulationillustrates the fact that the formulations are different. Importantly,the formulation described in Jorgensen 1991/Rosenfalck 1992 wasadministered in a divided dose with one half a dose administered in eachnostril. While this may be relatively easily performed in a clinicalresearch setting, under practical use conditions, this significantlycomplicates rescue treatment for non-medical caregivers in treating anepisode of severe hypoglycemia, as it would require administering twodoses of rescue medication. They also report sneezing in 50% of treatedpatients, a rate much higher than that observed (less than 2%) with theformulation described in this invention.

Intranasal administration to adults of the Jorgensen 1991 powderformulation is reported to show an increase in plasma glucoseconcentration in adults with hypoglycemia. In healthy adults withinsulin induced hypoglycemia, glucose levels increased after dosing toreach a plateau at about 45 minutes after dosing. In contrast, treatmentwith injected glucagon in this study resulted in glucose levels thatcontinued to increase from the time of administration for up to at least90 minutes (Hvidberg, 1994). In another study conducted with adults withtype 1 diabetes and insulin-induced hypoglycemia, glucose levelsincreased after dosing to reach a plateau at about 30 minutes afterintranasal dosing, compared to at least 90 minutes for injected glucagon(Rosenfalck, 1992).

In contrast to the glucose profile observed with the Jorgensen1991/Rosenfalck 1992 formulation, data generated with the powderformulation described in this invention show that plasma glucoseconcentrations continue to rise for at least 90 minutes after dosing(FIG. 7 , bottom line). As shown, this is comparable to the resultobtained with intramuscular glucagon over the same time period (FIG. 7 ,top line).

Intranasal administration to children with type 1 diabetes with inducedhypoglycemia of the Jorgensen 1991/Rosenfalck 1992 powder formulation isreported to increase plasma glucose concentration soon after dosing topeak levels 25-30 minutes post-dosing after which glucose levelsdecreased, compared with plasma glucose levels that continued to risefor at least 45 minutes after SC injection (Stenninger,1993). Peakpost-treatment glucagon values occurred at approximately 10 minutesafter intranasal dosing (Rosenfalck 1992; Stenninger 1993). In contrastto the glucose profile observed with the Jorgensen 1991/Rosenfalck 1992formulation, data generated in children (12-< 17 years) with the powderformulation described in this invention show plasma glucoseconcentrations that continue to rise for at least 60 minutes afterdosing (FIG. 8B). In addition, the peak plasma glucagon concentrationsdid not occur until approximately 20 minutes post-dosing (FIG. 8A).

Since episodes of severe hypoglycemia are unpredictable and could occurin insulin-using persons who are affected with nasal congestion, a studywas conducted to evaluate the PK and PD of the invented powderformulation in this situation. As described in Experiment 11 and shownin FIGS. 9A and 9B, the pharmacokinetics and pharmacodynamics resultingfrom treatment with the nasal powder of this invention are not adverselyaffected by nasal congestion. This supports the utility of thisinvention to treat episodes of severe hypoglycemia in people who may besuffering from nasal congestion. As was observed in people without nasalcongestion, the time to peak plasma glucagon levels was approximately 20minutes post-dosing. Data have never been reported to indicate whetheror not the Jorgensen 1991/Rosenfalck 1992 formulation can be used intreating a person with nasal congestion that could be seen in peoplesuffering from a common cold or seasonal allergic rhinitis.

Applicants believe that these differences in results arise from astructural difference between the claimed powder formulation and theJorgensen 1991/Rosenfalck 1992 powder. As discussed below,cosolubilization of the phospholipid and cyclodextrin componentsfollowed by drying and powder formation results in formation of somestructure that has a characteristic XRPD peak that is absent from eithermaterial alone. This peak is retained when glucagon is added to thecomposition. Without being bound by any particular theory, it isbelieved that the glucagon or glucagon analog associates with themicellar phospholipid in solution and maintains some association afterdrying, albeit without disrupting the structure formed by thephospholipid and cyclodextrin, and that this association better presentsthe glucagon for nasal absorption. Thus the claimed powder formulationis not simply an admixture of the three components, but rather containsunique physical structure that is detectable through X-ray powderdiffraction techniques.

This understanding of the structure of the formulation of the inventionis consistent with known information about the interaction of glucagonwith phospholipids surfactants. Glucagon forms complex structures at amolar ratio of about 55:1 phospholipid:glucagon (Epand & Sturtevant,1982). It has also been shown that glucagon can bind to 50 phospholipidmolecules, and that 20 of them are closely bound (Epand & Sturtevant,1981). Boesch et al (1980) and Brown et al (1981) report that theconformation of glucagon bound to various micellar lipids, includingDPC, is largely independent of the type of the lipid. The conformationis described as a well-defined, and predominantly extended. Thestoichiometry of the DPC:glucagon complex was reported as 40:1. Theyalso suggest the conformation of glucagon bound to micelles is verysimilar to that of glucagon bound to lipid bilayers.

The mole ratio of phospholipid (DPC, DDPC, LLPC, D8PC or DLPG): glucagonat a 10:10 weight ratio as in some of the preferred formulations of theinvention between 6:1 and 10:1, suggesting that the phospholipids aremost likely closely bound to glucagon in the intranasal powderformulations.

X ray powder diffraction studies on the powder formulation of thepresent invention clearly show the presence of a peaks likely to beassociated with a micellar or mesophase structure in the formulation.These peaks are characterized low diffraction angles (6.6 °2Θ for DPCand 7.3 °2Θ for DDPC). (FIG. 6 ) These same peaks are seen in samples inwhich glucagon is not included, and are referred to in this applicationas “XRPD mesopeaks.” This XRPD mesopeak is characteristic of the powderformulations of the invention.

FIG. 6 presents overlayed X-ray powder diffraction results forglucagon-DPC-ß-cyclodextrin (File 474320) andglucagon-DDPC-ß-cyclodextrin (File 407476) compositions. The diffractionpattern retains high angle peaks consistent with the presence ofcrystalline cyclodextrin (e.g the peak 61 at around 18-20 °-2θ), whichis not unexpected since the cyclodextrin is present in substantialexcess relative to phospholipid and glucagon. In addition, each patternhas a low diffraction angle peak at 6.6 and 7.3 °-2θ, respectively.These peaks are also present in samples without glucagon that are madeby solubilizing the phospholipid at micelle forming concentrations,adding the cyclodextrin and then drying the resulting solution. As usedin this application, the statement that “at least a portion of thepowder is present in a phase characterized by an XRPD mesopeak asdetermined by x-ray powder diffraction” indicates that the low anglepeaks are detectable in the x-ray powder diffractogram, and clearlydistinguishable from the noise of the measurement. In preferredembodiments, the size of the XRPD mesopeak (as determined by peakheight) is about 30% of the height of the peak at about 18-20 °-2θ (asin the pattern for File 407476 in FIG. 6 ) or greater, and may be aboutequal to the height of this peak (as in the pattern for File 474320 inFIG. 6 ) .

The formation of a phase characterized by an XRPD mesopeak has beenobserved in samples with and without glucagon, and using differentformulations and drying techniques. It has been observed that somevariation in shape and position of the XRPD mesopeak can occur dependenton the conditions of drying. For example, lyophilization of largeramounts that leads to a thicker frozen layer and longer drying times wasobserved in one experiment to lead to formation of two broadened andoverlapping XRPD mesopeaks.

A further benefit of the present invention is its usefulness as anemergency treatment, even under extreme environmental conditions,particularly cold conditions. The formulation of the invention remainsdirectly useable even when the temperature is below freezing and testshave shown that powder stored at -20° C. can be used directly withacceptable delivery and uptake of glucagon . In contrast, emergency kitsthat contain a liquid carrier for reconstitution of glucagon prior touse must be maintained above the freezing point of the carrier.Likewise, glucagon solutions must also be maintained above the freezingpoint of the solution, which will be at a higher temperature if asolvent such as DMSO is used to provide solution stability.

Method of Making the Compositions of the Invention

A further aspect of the present invention is a method for preparing apowder formulation having glucagon-activity comprising glucagon or aglucagon analog, a cyclodextrin, and a phospholipid surfactant whereinat least a portion of the powder is present in a phase characterized byan XRPD mesopeak as determined by x-ray powder diffraction. The methodcomprises the steps of

-   a. forming a first mixture of the glucagon and the surfactant in an    aqueous carrier, wherein the surfactant is present as a    concentration greater than or equal to the critical micelle    concentration;-   b. adding the cyclodextrin to the first mixture to form a second    mixture;-   c. drying the second mixture to form a solid formulation; and-   d. processing the solid formulation to produce a uniform powder,    said uniform powder including at least a portion the powder in a    phase characterized by an XRPD mesopeak.

Step a, forming a first mixture, can be accomplished by adding glucagonor a glucagon analog to a solvent, for example water, and then addingthe surfactant. Alternatively, the surfactant may be solubilized firstfollowed by addition of the glucagon or glucagon analog. The twocomponents of the mixture may also be individually solubilized and thencombined to form the first mixture.

Preferably, the solvent is acidified to a pH of 4 or less to enhance thesolubility of the glucagon. The acidification can be accomplished with amineral acid, such as HCl, phosphoric acid or sulfuric acid, or anorganic acid such as acetic acid, citric acid glycolic acid or lacticacid, or using a combination of a mineral acid and an organic acid. Inpreferred formulations, the acid is acetic acid.

The amount of solvent used to form the first mixture is sufficient tosolubilize the glucagon and phospholipid surfactant in the firstmixture. Excess solvent can be used, although large excesses increasethe amount of time and energy needed in the drying step and aretherefore not preferred.

The cyclodextrin can be added to the first mixture as a solid, or in asolvent, such as water to form the second mixture. Mixing can be carriedout by methods including static and dyamic mixing. Dynamic mixing can bedone by use of a blade inserted into the liquid, which is attached toshaft and rotated by a motor. Static mixing can be carried out byflowing the liquid through a tortuous path inside a static mixer. Thepresence of an air-water interface during mixing under high speed mixingconditions may result in foaming. The high speed mixing may also, inturn, result in destabilization of the protein due to the shear stress.In order to minimize foaming, and preferably eliminate it, low speedmixing conditions are preferred. In the case of dynamic mixing, thespeed is determined by the revolutions-per-minute (rpm) of the stirrer.Preferred rpm values are between 100 to 1000. In the case of staticmixing, the low shear conditions are obtained by selecting a pump thatallows for a non-laminar flow.

The second mixture is dried to remove the solvent (for example, water)and leave a solid product. Drying can be performed by freeze-drying,spray-drying, tray-drying or other techniques. The macroscopic physicalcharacteristics of the product will vary depending on the dryingtechnique, and may be in the form of a flaky solid from freeze drying ora dried solid cake. Regardless of the method used in drying, removal ofexcess water from the formulation has important effects on powdercharacteristics and stability.

Powders with excessive moisture content may be sticky and form clumpsresulting in a powder that is difficult to manipulate for filling of anadministration device. Importantly, the level of residual water contenthas a direct impact on the stability. In the case of glucagon, it iswell understood that the stability and physical characteristics areadversely affected by water. Specifically, in the presence of excesswater, glucagon forms amyloid fibrils that adversely affect thestability and toxicity profile of glucagon. (Pederson 2010). Because ofthis propensity to form amyloid fibrils, currently available glucagonproducts are supplied as a powder to be dissolved in water immediatelybefore use. Water can also adversely affect the stability of glucagondue to hydrolysis, oxidation and deamidation. To this end, datagenerated with the formulations of the invention indicate that residualmoisture content levels in excess of 5% in the bulk powder results inreduced stability compared to powder with residual water content below5%. Suitable powders for nasal administration therefore typically haveresidual water content levels below 5%.

Suitable powders for nasal administration typically have particle sizedistributions such that most particles are greater than approximately 10microns and smaller than approximately 1000 µm. Preferably, particlesize distributions will be such that the D10 falls within the range of3-75 µm, the D50 falls within the range of 15 to 450 µm, and the D90falls within the range of 80-1000 µm, with a span of 1.5 to 15.

Suitable powders for nasal administration require physicalcharacteristics that permit adequate flowability to allow for fillingthem into a nasal discharge device. Flowability is determined by variousparameters including particle size, shape, density, surface texture,surface area, density, cohesion, adhesion, elasticity, porosity,hygroscopicity, and friability.

Powders with the appropriate particle size and flowabilitycharacteristics may be produced by processing the bulk power to removeparticles that are too small or too large. Methods of processing thebulk powder to remove the particles that are too small or too large mayinclude milling the bulk powder to break up larger particles and sievingto isolate the particles of the desired particle size range. Variousmethods of sieving may be performed including throw-action sieving,horizontal sieving, tapping sieving, super-sonic sieving and aircircular jet sieving. Sieves may be used as single sieves of a fixednominal aperture or the bulk powder may be processed through a series ofsieves of progressively smaller apertures to obtain the desired particlesize distribution. Sieves may be woven wire mesh sieves with nominalapertures ranging from 25 - 1000 µm.

Spray pattern and plume geometry resulting from administration of thepowder with a nasal powder administration device are importantcharacteristics that govern the surface area upon which the nasal powderis deposited within the nasal cavity. Suitable spray patterncharacteristics for the invention include a minimum diameter (Dmin) of5 - 25 mm, a maximum diameter (Dmax) of 10 to 60 mm, and an ovalityratio of 0.5 to 6. Specific spray patterns observed for formulations ofthe invention are summarized in the following Table

Spray Pattern Dmin (mm) Dmax (mm) Ovality ratio (min/max) (mean)(min/max) (mean) (min/max) (mean) 10.7-21.9 13.9 14.5-42.9 23.6 1.1-3.61.7

Suitable plume geometry characteristics for the invention include aspray angle falling within the range of 20 to 75° and a plume widthfalling within the range of 10 to 50 mm. The following table summarizesthe plume geometry information for multiple lots of powder in accordancewith the invention.

Plume Geometry Spray Angle (°) Plume Width (mm) (min/max) (mean)(min/max) (mean) 28.31-65.2 44.8 15.2-38.4 25.0

Administration of and Applicators for the Composition of the Invention

While any methodology for introducing the powder into a user’s nose maybe used in the method of the invention, the powder composition of theinvention is suitably provided in a purpose-designed nasal applicatorthat maintains the powder in a clean, dry and usable state until use,and then delivers the powder to the nasal mucosa of a user. Suchapplicators are known in the art, and generally have a powderformulation reservoir, and a powder formulation contained within thereservoir, and a mechanism for expelling the powder formulation from thereservoir through a nozzle receivable within a nostril.

The applicator is selected to be able to provide sufficient powderformulation in a single insufflation/administration to provide atherapeutic dose. Larger reservoirs and delivery capacity are requiredfor powders with lower percentages of glucagon or glucagon analog in theformulation, while smaller reservoirs and delivery capacity can be usedwith higher percentage formulations.

Specific suitable delivery devices are disclosed in U.S. Pats. Nos.6,398,074 and 6,938,798, which are incorporated herein by reference.FIG. 10 is taken from the ‘074 patent to illustrate a suitable device.In FIG. 10 , reservoir 810 contains a single dose of a formulation inaccordance with the present invention. The reservoir 810 has an airinlet 811 and a product outlet 815. A product retention device 812and/or 813 is disposed in the air inlet 811 to keep the product in thereservoir 810 until dispensation of the product. The product outlet 815is blocked, preferably in a sealed fashion, by a closing ball 816 whichis removed from its blocking position by the flow of air when theproduct is being dispensed.

When a user actuates the device, he exerts a pressure on the plunger 825in such a way that the piston 821 compresses the air contained in thechamber 822 of the air blast 820. Since grid 812 is permeable to air,the compression of the air in chamber 822 is transmitted to thereservoir 810 and consequently is applied to the closing ball 816 whichis blocking the product outlet 815. The dimensions of the closing ball816 and its fixing at the reservoir product outlet 815 are such that theball 816 is removed from its blocking position, when a minimumpredetermined pressure is created through the reservoir 810 in said airblast 820. Hence, when this minimum pressure is reached, the ball issuddenly moved towards the outlet channel 840 of the device and the flowof air created by the air blast 820 expels all of the dose contained inthe reservoir 810. The pre-compression created by this closing ball 816ensures that when it is removed from its blocking position, the energyaccumulated in the hand of the user is such that the piston 821 integralwith the plunger 825 is propelled within the chamber 822 therebycreating a powerful air flow, that is to say an air flow suitable tofinely spray the dose of product and notably to get rid of any productagglomerates if it is a powder product.

Another example of an applicator device suitable for use in combinationwith the powder composition of the invention is that disclosed in U.S.Pat. Publication No. 20110045088, which is incorporated herein byreference. The device shown in U.S. Pat. No. 7,722,566 could also beused, particularly as shown in FIGS. 1 and 7 thereof, sinceadministration into both nostrils is not required using the powder ofthe invention.

Still further examples of applicator devices for nasal administration ofa powder composition are known from WO2014004400, and U.S. Pat. No.5,702,362, which are incorporated herein by reference.

EXPERIMENTAL Example 1

Glucagon, DPC and α-CD, β-CD, or HP-ß-CD were dissolved in either a 0.01N or a 0.1 N HCl solution. Formulations were also prepared with either 1M acetic acid or 0.5 M acetic acid. The weight ratio ofglucagon:DPC:cyclodextrin ranged from 5:10:75 to 10:20:70. In twoseparate experiments, either sodium citrate or citric acid was added asan additive. The lyophilized powder was packaged into a device fordelivery to the nostril. The powder was delivered to Beagle dogsintranasally at a dose of 500 µg, 750 µg or 1000 µg. The powder wasadministered to either 3 or 6 dogs per group. Plasma glucoseconcentration was measured by using a glucometer. The plasma glucoseconcentrations prior to administration (0 min), and 5, 10, 20, 30, 40and 60 minutes after nasal administration are shown in Table 1. In Table1, Ratio refers to the ratio of glucagon:DPC:cyclodextrin, or to theratio of glucagon:DPC:cyclodextrin:additive.

TABLE 1 Average plasma glucose concentrations (mmol/L) in the Beagle dogafter intranasal administration of glucagon:DPC:CD compositions Ratio CDDose per nostril (µg) Nostrils Acid Additive Plasma glucoseconcentration (mmol/L) 0 min 5 min 10 min 20 min 30 min 40 min 60 min5:25:70 α-CD 500 1 0.1 N HCl None 3.7 3.4 6.1 6.3 3.6 2.4 3.4 5:25:70α-CD 750 1 0.1 N HCl None 3.7 3.7 4.4 4.7 6.9 5.0 3.8 5:25:70 α-CD 10001 0.1 N HCl None 3.7 3.9 4.8 7.9 5.8 3.5 3.2 5:25:70 α-CD 500 2 0.1 NHCl None 3.3 3.3 3.7 4.1 4.0 3.2 3.5 5:40:55 α-CD 500 1 0.1 N HCl None3.7 3.6 3.8 3.8 3.5 4.2 4.2 5:40:55 α-CD 1000 1 0.1 N HCl None 3.6 3.64.3 6.7 5.1 3.8 3.2 5:25:70 α-CD 750 1 0.1 N HCl None 3.7 3.5 4.2 4.34.3 4.1 4.0 10:30:60 HP-β-CD 750 1 0.1 N HCl None 3.7 4.2 5.2 5.4 5.04.4 3.9 10:30:60 HP-β-CD 750 1 0.1 N HCl None 3.4 4.2 5.2 5.1 4.9 4.03.7 10:70:10:10 α-CD 750 1 0.1 N HCl Citric acid 3.6 5.2 6.0 6.2 5.9 4.53.3 10:70:10:10 α-CD 750 1 0.1 N HCl Sodium citrate 3.7 5.4 6.6 6.3 5.54.4 3.9 10:10:80 α-CD 750 1 0.1 N HCl None 3.8 5.6 7.3 9.0 8.0 6.0 3.110:10:80 α-CD 750 1 0.1 N HCl None 4.0 4.1 5.1 5.6 6.1 6.1 5.7 5:10:85α-CD 750 1 0.1 N HCl None 3.9 3.8 4.4 4.2 4.1 4.0 4.0 10:10:80 ß-CD 7501 0.1 N HCl None 3.8 4.7 7.0 7.7 8.1 6.7 5.2 10:10:80 α-CD 600 1 0.01 NHCl None 4.0 4.3 5.7 5.6 5.5 4.6 4.0 10:10:80 ß-CD 750 1 0.01 N HCl None4.5 5.1 6.6 7.2 6.8 5.5 4.8 10:10:80 ß-CD 600 1 0.01 N HCl None 4.0 4.35.4 4.9 4.9 3.6 3.7 10:10:80 ß-CD 1000 1 0.01 N HCl None 4.4 7.4 8.7 8.88.1 5.9 4.6 10:10:80 ß-CD 750 1 0.01 N HCl None 4.4 5.4 6.8 6.1 5.1 4.04.0 10:10:80 ß-CD 500 1 0.01 N HCl None 4.4 5.4 7.3 6.6 6.1 4.9 4.310:10:80 ß-CD 500 1 1 M acetic acid None 4.40 5.08 6.00 5.80 5.17 4.654.53 10:10:80 ß-CD 1000 1 1 M acetic acid None 3.80 5.08 8.22 9.74 10.07.96 6.44 10:10:80 ß-CD 2000 1 1 M acetic acid None 4.23 6.50 10.2 12.312.7 11.2 8.85

All compositions (prepared with different concentrations of the acid,different acids, different ratios of the three ingredients, in thepresence of citric acid or sodium citrate, different doses, delivered toone nostril or to both nostrils) show an increase in plasma glucoseconcentration for up to between 10 and 30 minutes, followed by adecrease at 40 minutes, and followed by a further decrease at 60minutes. As an example, results from a 10:10:80 composition with β-CDand 0.01 N HCl administered at a 1000 µg dose to one nostril are shownin FIG. 1 .

Example 2

Glucagon, DDPC, and α-CD, ß-CD, or hydroxylpropyl-ß-CD were dissolved ineither a 0.01 N or a 0.1 N HCl solution. The weight ratio ofglucagon:DDPC:cyclodextrin ranged from 5:10:75 to 10:20:70. In onestudy, sodium citrate was also added. The powder was packaged into adevice for delivery to the nostril. The powder was delivered to Beagledogs intranasally at a dose of 500 µg, 750 µg or 1000 µg. The powder wasadministered to either 3 or 6 dogs per group. Plasma glucoseconcentration was measured by using a glucometer. The results are shownin the following table. The plasma glucose concentrations prior toadministration (0 min), and 5, 10, 20, 30, 40 and 60 minutes after nasaladministration are shown in Table 2. In Table 2, Ratio refers to theratio of glucagon:DDPC:cyclodextrin, or to the ratio ofglucagon:DDPC:cyclodextrin:additive.

TABLE 2 Average plasma glucose concentrations (mmol/L) in the Beagle dogafter intranasal administration of glucagon:DDPC: CD compositions RatioCD Dose per nostril (µg) Nostrils Acid Additive Plasma glucoseconcentration (mmol/L) 0 min 5 min 10 min 20 min 30 min 40 min 60 min5:10:85 α-CD 500 1 0.1 N HCl None 5:10:85, α-CD, 500 µg, 1 nostril 3.83.7 4.6 6.0 6.3 4.3 3.4 5:10:85 α-CD 750 1 0.1 N HCl None 5:10:85, α-CD,750 µg, 1 nostril 3.6 3.8 7.8 7.6 8.3 4.7 3.3 5:10:85 α-CD 1000 1 0.1 NHCl None 5:10:85, α-CD, 1000 µg, 1 nostril 3.8 4.0 4.7 7.4 7.1 4.3 3.85:10:85 α-CD 500 2 0.1 N HCl None 5:10:85, α-CD, 500 µg per nostril, 2nostrils 3.4 3.1 3.4 5.3 5.0 3.2 3.2 5:51:44 α-CD 500 1 0.1 N HCl None5:51:44, α-CD, 500 µg, 1 nostril 3.3 3.2 4.0 4.6 4.3 3.6 3.9 5:51:44α-CD 500 2 0.1 N HCl None 5:51:44, α -CD, 500 µg per nostril, 2 nostrils3.4 2.8 4.0 4.8 3.8 3.6 3.7 5:10:85 α-CD 750 1 0.1 N HCl None 5:10:85, α-CD, 750 µg, 1 nostril 3.7 5.1 6.4 7.5 7.9 7.3 6.3 10:41:49 HP-ß-CD 7501 0.1 N HCl None 10:41:49, HP-β-CD, 750 µg, 1 nostril 3.7 3.5 4.1 4.04.0 3.8 3.9 10:41:49 HP-ß-CD 750 1 0.1 N HCl None 10:41:49, HP-β-CD, 750µg, 1 nostril 3.7 4.6 5.6 5.5 4.9 4.1 3.7 10:25:55:10 α-CD 750 1 0.1 NHCl Sodium Citrate 10:25:55:10, α-CD, sodium citrate, 750 µg, 1 nostril3.6 4.8 6.3 6.3 5.5 4.6 4.0 10:20:70 α-CD 750 1 0.1 N None 10:20:70,α-CD, 750 µg, 3.5 3.9 4.4 4.3 4.4 4.0 3.9 HCl 1 nostril 10:10:80 α-CD750 1 0.1 N HCl None 10:10:80, α-CD, 750 µg, 1 nostril 3.5 5.3 6.7 7.86.7 5.3 3.7 10:10:80 α-CD 750 1 0.1 N HCl None 10:10:80, α-CD, 750 µg, 1nostril 3.8 4.9 5.5 6.9 5.6 4.4 4.0 5:10:85 α-CD 750 1 0.1 N HCl None5:10:85, α-CD, 750 µg, 1 nostril 4.0 4.3 5.2 5.6 5.6 5.6 5.2 10:10:80ß-CD 750 1 0.1 N HCl None 10:10:80, β-CD, 750 µg, 1 nostril 3.6 4.7 6.86.5 6.1 4.6 4.0 10:10:80 ß-CD 700 1 0.1 N HCl None 10:10:80, β-CD, 700µg, 1 nostril 4.0 5.5 7.1 6.9 7.0 5.2 4.3 10:10:80 α-CD 750 1 0.01 N HClNone 10:10:80, α-CD, 750 µg, 1 nostril, 0.01 N HCl 4.3 4.7 6.0 6.4 6.55.1 4.5 10:10:80 ß-CD 1000 1 0.01 N HCl None 10:10:80, ß-CD, 1000 µg, 1nostril, 0.01 N HCl 4.2 6.4 8.7 8.5 7.5 4.6 3.6 10:10:80 ß-CD 750 1 0.01N HCl None 10:10:80, ß-CD, 750 µg, 1 nostril, 0.01 N HCl 4.5 6.1 6.4 6.45.8 4.4 3.9 10:10:80 ß-CD 500 1 0.01 N HCl None 10:10:80, ß-CD, 500 µg,1 nostril, 0.01 N HCl 4.4 5.8 6.8 5.4 4.6 4.0 4.1

All compositions (prepared with different concentrations of the acid,different cyclodextrins, different ratios, different doses, delivered toeither one nostril or to both nostrils) show an increase in plasmaglucose concentration for up to between 10 and 30 minutes, followed by adecrease at 40 minutes, and further followed by a decrease at 60minutes. As an example, results from a 10:10:80 composition with β-CDadministered at a 1000 µg dose delivered to one nostril are shown inFIG. 2 .

Example 3

Glucagon, LLPC and β-CD were dissolved in either a 0.01 N or a 0.1 N HClsolution. The weight ratio of glucagon:LLPC: β-CD was 10:10:80. Thepowder was packaged into a device for delivery to one nostril. Thepowder was delivered to Beagle dogs intranasally either at a dose of 750µg or 1000 µg. The powder was administered to 6 dogs per group. Plasmaglucose concentration was measured by using a glucose strip. The plasmaglucose concentrations prior to administration (0 min), and 5, 10, 20,30, 40 and 60 minutes after nasal administration are shown in Table 3.

TABLE 3 Average plasma glucose concentrations (mmol/L) in the Beagle dogafter intranasal administration of glucagon:LLPC: CD compositions Dosein µg Acid Plasma Glucose Concentration (mmol/L) 0 min 5 min 10 min 20min 30 min 40 min 60 min 750 0.1 N HCl 3.8 4.8 6.6 6.9 6.1 4.4 4.0 7500.1 N HCl 4.2 4.5 5.8 5.7 5.7 5.2 4.6 1000 0.01 N HCl 4.5 6.6 7.6 7.37.4 5.4 4.2

All compositions (different doses, delivered to one nostril show anincrease in plasma glucose concentration for up to between 10 and 20minutes, followed by a decrease at either 30 or 40 minutes, and furtherfollowed by a decrease at 40 or 60 minutes. As an example, results fromthe 10:10:80 composition with β-CD administered at a 1000 dose to onenostril is shown in FIG. 3 .

Example 4

Glucagon, D8PC and β-CD were dissolved in either a 0.01 N or a 0.1 N HClsolution. The weight ratio of glucagon:D8PC: β-CD was 10:10:80. Thepowder was packaged into a device for delivery to the nostril. Thepowder was delivered to Beagle dogs intranasally. The powder wasadministered to 6 dogs per group. Plasma glucose concentration wasmeasured by using a glucose strip. The plasma glucose concentrationsprior to administration (0 min), and 5, 10, 20, 30, 40 and 60 minutesafter nasal administration are shown in Table 4.

TABLE 4 Average plasma glucose concentrations (mmol/L) in the Beagle dogafter intranasal administration of glucagon:D8PC: CD compositions AcidPlasma Glucose Concentration (mmol/L) 0 min 5 min 10 min 20 min 30 min40 min 60 min 0.1 N HCl 4.0 5.4 7.4 7.7 7.4 6.0 4.8 0.01 N HCl 3.5 4.04.8 4.5 4.3 4.0 3.9 0.01 N HCl 4.1 4.7 6.3 5.7 5.0 3.9 3.9 0.01 N HCl3.7 4.3 5.4 5.4 5.3 4.7 4.4

All compositions (prepared with different concentrations of the aciddelivered to one nostril) show an increase in plasma glucoseconcentration for up to between 10 and 20 minutes, followed by adecrease at 30 or 40 minutes, and further followed by a decrease at 40or 60 minutes. As an example, results from the 10:10:80 composition withβ-CD administered at a 750 µg dose to one nostril are shown in FIG. 4 .

Example 5

Glucagon, DLPG and α-CD were dissolved in a 0.1 N HCl solution. Theweight ratio of glucagon:DLPG: α-CD was either 5:25:70 or 5:54:41.Separately, glucagon, DLPG and ß-CD were dissolved in 0.1 N HCl solutionat a weight ratio of 10:10:80. The resultant solutions were lyophilizedto produce a powder. The powder was packaged into a device for deliveryto the nostril. The powder was delivered to either one nostril or bothnostrils of Beagle dogs. The powder was administered to either 3 or 6dogs. Plasma glucose concentration was measured by using a glucosestrip. The results are shown in the following table. The weight ratio ofglucagon:DLPG: α-CD (or, ß-CD), dose of glucagon per nostril, whetherthe powder was delivered to one or both nostrils are shown in the table.The plasma glucose concentrations prior to administration (0 min), and5, 10, 20, 30, 40 and 60 minutes after nasal administration are shown inTable 5.

TABLE 5 Average plasma glucose concentrations (mmol/L) in the Beagle dogafter intranasal administration of glucagon:DLPG: CD compositions RatioCD Dose per Nostril in µg Nostrils Plasma Glucose Concentration (mmol/L)0 min 5 min 10 min 20 min 30 min 40 min 60 min 5:25:70 α-CD 500 1 3.93.9 4.1 4.0 4.6 4.4 4.1 5:25:70 α-CD 750 1 3.8 3.5 5.3 6.6 7.2 4.6 3.65:25:70 α-CD 1000 1 3.4 4.0 4.4 5.7 4.4 3.7 4.3 5:25:70 α-CD 500 2 3.83.1 3.1 3.2 4.3 4.1 4.0 5:54:41 α-CD 500 1 3.4 3.0 3.2 4.2 3.2 4.3 4.010:10:80 β-CD 750 1 4.2 4.7 6.1 6.0 5.5 4.6 4.0

All compositions (prepared with different cyclodextrins, differentratios, different doses, delivered to either one nostril or to bothnostrils) show an increase in plasma glucose concentration for up tobetween 10 and 30 minutes, followed by a decrease at 40 minutes, andfurther followed by a decrease at 60 minutes. As an example results fromthe 5:25:70 composition with α-CD administered at a 750 µg dose to onenostril, which resulted in the highest plasma glucose concentration, areshown in FIG. 5 .

Example 6

X-ray powder diffraction was used to determine the structure of theglucagon-DPC-ß-cyclodextrin and glucagon-DDPC-ß-cyclodextrincompositions. They exhibit a peak at low angles (6.6 °2Θ for DPC and 7.3°2Θ for DDPC) indicating a mesophase (FIG. 6 ). These peaks are absentfor glucagon because it is an amorphous powder. They are also absent inß-cyclodextrin which exhibits a characteristic crystalline form. Furtherthey are absent in the surfactant, DPC. These peaks are present inmixtures of DDPC (or DPC) and ß-cyclodextrin, in the absence ofglucagon. The compositions of the current invention are characterized bya mesophase detectable through these low diffraction angle peaks.

Example 7

The volume-weighted distribution profile is used to calculate D₁₀, D₅₀,and D₉₀ are presented in Table 6.

TABLE 6 Diameter of 10% of the particles (D₁₀), 50% of the particles(D₅₀), and 90% of the particles (D₉₀) Surfactant Acid D₁₀ (µm) D₅₀ (µm)D90 (µm) DDPC 0.1 N HCl 6.063 11.77 21.86 DPC 0.1 N HCl 6.63 66.51 426DPC with sonication 0.1 N HCl 3.6 10.68 21.77 D8PC 0.1 N HCl 7.926 17.9735.34 LLPC 0.1 N HCl 8.031 15.31 28.89 DPC 1 M acetic acid 6.867 92.480537.471

The particle size analysis shows that a minority of the particles in thesix different powder formulations have an effective diameter rangingfrom 3.6 to 8.031 µm. The D₁₀ results show that greater than 90% of theparticles in the powder delivered to the nostril cannot be inhaled.

Example 8

A powder formulation was prepared using the methods described in Methodof Preparation. The composition was 10:10:80 glucagon:DPC:ß-cyclodextrinprepared using 1 M acetic acid. Ten milligrams of the powder contained 1mg of glucagon. Ten milligrams of the powder formulation were packagedinto each of ten devices. The powder delivered upon actuation of thedevice was collected. The weights of the delivered powder from the tendevices are shown in Table 7, as Delivered Dose (mg). The Uniformity ofDelivered Dose (%) from the ten devices, also shown in Table 7, wascalculated by multiplying by 100 the ratio of the amount of glucagon ineach of the ten delivered powders to the amount of glucagon in 10milligrams of the powder before it was filled into each of the tendevices.

TABLE 7 Delivered dose and uniformity of the delivered dose DeviceNumber Uniformity of Delivered Dose (%) Delivered Dose, mg 1 97.5 9.3 297.0 9.2 3 98.9 9.7 4 102.7 9.8 5 100.3 9.7 6 92.3 9.0 7 100.2 9.6 896.9 9.2 9 94.8 9.2 10 96.8 9.2

Example 9

A multiple center, randomized, two-way, crossover Phase III study inwhich patients with type 1 diabetes (T1D, n=75) were enrolled toevaluate the effectiveness and safety of a single dose of intranasallyadministered glucagon (the powder that is the subject of this invention)compared to glucagon administered by IM injection followinginsulin-induced hypoglycemia was conducted. The primary endpoint in thisstudy was the proportion of patients who achieved an increase in bloodglucose levels to ≥ 70 mg/dL within 30 minutes of treatment withglucagon or a 20 mg/mL increment in blood glucose within 30 minutes oftreatment with glucagon. The protocol used insulin to reduce bloodglucose levels to the hypoglycemic range (target nadir of <50 mg/dL).

Statistical analysis of the glycemic response to treatment with thenasal powder showed that the nasal powder was non-inferior to injectedglucagon in treating insulin-induced hypoglycemia. The glucose responsecurve, presented in FIG. 7 , shows that glucose levels increasedsubstantially in both the nasal and injected treated groups and thatblood glucose levels increased to within the normal range in mostsubjects in both groups by about 15 minutes post-dosing.

Example 10 - Amg103

AMG 103 was a study in children with type 1 diabetes, aged 4-<17 years.Induction of severe hypoglycemia in this population is not permitted bypediatric IRBs but insulin was used if necessary to normalize bloodglucose to a target of <80 mg/dL (4.4 mmol/L) prior to dosing withglucagon.

Children visited the study facility twice. At the first visit, 12children aged 12 to < 17 years were randomized to glucagon by IMinjection (dose rate according to the manufacturer’s labeling) or to aglucagon powder formulation that is the subject of this invention(10:10:80 by weight). At the second visit, subjects received thealternative treatment. For children in the 4 to <8 years and 8 to <12year age groups, there were 18 children per group. Within each of theseage groups, children were randomized 2:1 to receive either two doses ofintranasal glucagon or a single injection of glucagon IM. For thechildren receiving the IN glucagon, they were randomized to receive 2 or3 mg on the first visit and the alternative dose level on the secondvisit. Study participants and the study site were blinded to the doselevel.

Results from the children aged 12-< 17 are provided as an example ofwhat was seen in children dosed with nasal glucagon powder. FIG. 8Aprovides the glucagon PK curve while FIG. 8B provides the glucoseprofile. The data generated in this study indicate that nasal powderglucagon resulted in a glucose response that was no different than thatobserved after an injection of glucagon.

Example 11 - Effect of Nasal Congestion

The powder of this invention consisting of glucagon:DPC and betacyclodextrins in ratios of 10:10:80 by weight, was tested in subjectswith common cold with and without concomitant administration of nasaldecongestant in a study to investigate the safety and PK/PD of a 3 mgdose of IN glucagon in male and female subjects. This was a singlecenter, single dose, open-label, repeated measures, parallel designstudy. All thirty-six (36) subjects received a single 3 mg dose ofglucagon by intranasal route, in the morning after a 10-hour overnightfast. Cohort 1 (18 subjects) was scheduled for two periods. DuringPeriod 1, the subjects had nasal congestion and/or nasal dischargeassociated with a common cold and during Period 2, the subjects hadrecovered from the common cold and had been symptom free for at least 2days. In Cohort 2 (Subjects #019 to 036), the subjects were scheduledfor only Period 1. After presenting with nasal congestion and/or nasaldischarge associated with a common cold, these subjects were pretreatedwith a nasal decongestant prior to receiving a single IN dose ofglucagon.

Measurements of peak nasal inspiratory air flow provided an objectivemeasurement of the nasal congestion and confirmed the nasal congestionassociated with common cold as well as the intended therapeutic effectof oxymetazoline.

The study drug was well tolerated and there were no serious adverseevents or deaths during this study.

The glucagon and glucose responses after administration of the powderare presented in FIGS. 9A and 9B. Plasma glucagon concentrations (FIG.9A) increased substantially above baseline with mean peak concentrations(C_(max)) of 1198.4, 868.0 and 801.5 pg/mL for “AMG504-1 + Common cold”,“Common cold + Decongestant”, and “No Cold Symptoms”, respectively.Median time to peak concentrations (t_(max)) was 20 minutes post dosefor all treatment groups. The estimated AUC_(0-t) for ‘AMG504-1 + CommonCold’ was higher than the other two treatment groups (1198.4 vs. 1038.0and 797.5).

Blood glucose levels (FIG. 9B) in all three groups began to increase by5 minutes post-dosing indicating nasal congestion, with or withoutconcomitant administration of a nasal decongestant, did not have aneffect on the onset the glycemic response. Overall, the glucose profileafter administration of intranasal glucagon was comparable regardless ofthe presence of common cold or the administration of a decongestant insubjects with common cold.

The results of this study indicate the PK or PD of the powderadministered intranasally is not significantly affected by nasalcongestion associated with common cold, with or without concomitantadministration of a nasal decongestant. This is very important becausepeople with diabetes who take insulin are at risk of experiencing severehypoglycemia at any time, including when suffering from allergies or acommon cold. As such, an intranasal glucagon intended for treatment ofsevere hypoglycemia must also be effective in the presence of nasalcongestion.

Example 12

Biocompatibility, safety and tolerability of the compositions of thisinvention were evaluated in a series of studies conducted in rats, dogsand rabbits. Subchronic and acute toxicity were evaluated. Table 8 showsfindings from these studies. The studies show that the compositions ofthe current invention are well tolerated with no adverse effects.

TABLE 8 Subchronic and acute toxicity studies in rats, dogs and rabbitsStudy Type Species Test Articles & Dosage Key findings 28 day subchronictoxicology Dog Saline, placebo powder, AMG504-1 at 2 and 4 mg/dog/dayfor 28 days Other than transient (i.e., < 30 seconds) snorting andsalivation immediately post-dosing, no adverse clinical signs. Noadverse gross necropsy findings or treatment-related effects on BW, foodconsumption, clinical chemistry, hematology, EKG or organ weights.Minimal to moderate fully reversible atrophy/degeneration of theolfactory epithelium. No microscopic test article-related findings uponhistological examination of all tissues. 28 day subchronic toxicologyRat Saline, placebo liquid, AMG504-1 ingredients in solution at 0.1 and0.2 mg/rat/day for 28 days No adverse clinical signs or gross necropsyfindings. No treatment-related effects on BW, food consumption, clinicalchemistry, hematology, EKG or organ weights. Minimal to moderate fullyreversible erosion/ulceration of olfactory epithelium in high dosegroup. No microscopic test article-related findings upon histologicalexamination of all tissues. Acute toxicology Rat Air placebo control,AMG504-1 at 0.5 mg intratracheally No adverse clinical, macroscopic ormicroscopic findings Acute toxicology Rabbit 30 mg drug productadministered directly in eye Well tolerated, with minimal ocularirritation limited to slight erythema and edema localized to theconjunctiva and palpebral membrane.

References (All of Which Are Incorporated Herein by Reference)

C. Boesch, L.R. Brown, and K. Wuethrich. Physicochemicalcharacterization of glucagon-containing lipid micelles. Biochim.Biophys. Acta 603: 298-312, 1980.

L.R. Brown, C. Boesch and K. Wuthrich. Location and orientation relativeto the micelle surface for glucagon in mixed micelles withdodecylphosphocholine: EPR and NMR studies. Biochim. Biophys. Acta 642:296, 1981

S. Carstens and M. Sprehn. Prehospital treatment of severehypoglycaemia: A comparision of intramuscular glucagon and intravenousglucose. Prehospital and Disaster Medicine 13: 44-50, 1998

Chabenne et al., MOLECULAR METABOLISM 3 (2014) 293-300.

P.E. Cryer. Hypoglycaemia: The limiting factor in the glycaemicmanagement of type I and type II diabetes. Diabetologia 45: 937-948,2002.

P.E. Cryer. Hypoglycemia in diabetes. American Diabetes Association.2009.

R. Curran. A milestone change in practice. A call for widespreadapplication of intranasal medication delivery in the prehospitalenvironment. www.emsworld.com. 2007

I.J. Deary. Symptoms of hypoglycaemia and effects on mental performanceand emotions. In: Hypoglycaemia in Clinical Diabetes. Second Edition.Eds. B.M. Frier and M. Fisher. 2007.

H. Dodziuk. Cyclodextrins and their complexes. Wiley-VCH Verlag, Berlin,2006.

K. Endo, S. Amikawa, A. Matsumoto, N. Sahashi and S. Onoue.Erythritol-based dry powder of glucagon for pulmonary administration.Int. J. Pharm. 290: 63-71, 2005.

R. M. Epand and J.M. Sturtevant. A calorimetric study ofpeptide-phospholipid interactions: Theglucagon-dimyristoylphosphatidylcholine complex. Biochemistry 20:4603-4606, 1981. R.M. Epand and J.M. Sturtevant. Studies on theinteraction of glucagon with phospholipids. Biophys. J. 37: 163-164,1982.

J. Filipovic-Grcic and A. Hafner. Nasal powder drug delivery. Chapter5.7, p. 651-681, In: Pharmaceutical Manufacturing Handbook: Productionand Processes, Edited by S.C. Gad, Wiley-Interscience, New York, 2008,pp. 1384.

L. Freychet, N. Desplanque, P. Zirinis, S.W. Rizkalla, A. Basdevant, G.Tchobroutsky and G. Slama. Effect of intranasal glucagon on bloodglucose levels in healthy subjects and hypoglycaemic patients withinsulin-dependent diabetes. The Lancet. pp. 1364-1366, Jun. 18, 1988

Glucagon for injection (rDNA origin). Package Insert. Eli Lilly & Co.2005.

G. Harris, A. Diment, M. Sulway and M. Wilkinson. Glucagonadministration -underevaluated and undertaught. Practical Diabetes Int.18: 22-25, 2001.

M.A. Howell and H.R. Guly. A comparison of glucagon and glucose inprehospital hypoglycaemia. J. Accid. Emerg. Med. 14: 30-32 (1997).

A. Hvidberg, R. Djurup and J. Hilsted. Glucose recovery after intranasalglucagon during hypoglycaemia in man. Eur. J. Clin. Pharmacol. 46:15-17, 1994.

IDF Diabetes Atlas, International Diabetes Foundation, Fourth Edition,2009.

ISMP Canada Safety Bulletin. Administration of product-specific diluentwithout medication. Institute for Safe Medication Practices Canada.10(7): 1-3, 2010.

K.K. Jain. Drug Delivery Systems. Methods in Molecular Biology. Vol.437. pp. 8-9, Humana Press, Totowa, NJ, 2008.

S. Jorgensen, A.R. Sorensen, L.L. Kimer and N. Mygind. A powderyformulation of glucagon for nasal delivery- a phase 1 study. Diabetes 40[Suppl A]:2190, 1991

N.C. Kaarsholm. Stabilized aqueous glucagon solutions comprisingdetergents. Patent numbers WO9947160, EP19990906095, 1999.

N.C. Kaarsholm. Stabilized aqueous peptide solutions. U.S. Pat. No.6,384,016, 1999a.

D. Lichtenberg. Liposomes as a model for solubilization andreconstitution of membranes. Chapter 13. pp. 199-218. In: Handbook ofnonmedical applications of liposomes. Models for biological phenomena.Vol II, Ed. Y. Barelholz and D.D. Lasic, CRC Press, Boca Raton FL. pp.379, 1996

Marsh, D. Handbook of lipid bilayers. CRC Press, Boca Raton, FL, 387pp.,1990.

L. Matilainen, K.L. Larsen, R. Wimmer, P. Keski-Rahkonen, S. Auriola, T.Jarvinen and P. Jarho. The effect of cyclodextrins on chemical andphysical stability of glucagon and characterization of glucagon/gamma-CDinclusion complexes. J. Pharm. Sci. 97: 2720-2729, 2008.

L. Matilainen, S. L.. Maunu, J. Pajander, S. Auriola. I. Laaskelainen,K.L. Larsen, T. Jarvinen and P. Jarho. The stability and dissolutionproperties of solid glucagon/gamma-cyclodextrin powder. Eur. J. Pharm.Sci. 36: 412-420, 2009.

S. Onoue, K. Yamamoto, Y. Kawabata, M. Hirose, T. Mizumoto and S.Yamada. Novel dry powder inhaler formulation of glucagon with additionof citric acid for enhanced pulmonary delivery. Int. J. Pharm. 382:144-150, 2009.

R.P. Oomen and H. Kaplan. Binding of glucagon to lipid bilayers.Biochem. Cell Biol. 68: 284-291, 1990.

J.E. Sondergaard Pederson. The nature of amyloid-like glucagon fibrils.J. Diab. Science Tech. 4: 1357-1367. 2010.

A.E. Pontiroli, M. Alberetto and G. Pozza. British Medical Journal. 287:462-463, 1983. A.E. Pontiroli, M. Alberetto, A. Calderara, E. Pajettaand G. Pozza. Nasal administration of glucagon and human calcitonin tohealthy subjects: a comparison of powders and spray solutions and ofdifferent enhancing agents. Eur. J. Clin. Pharmacol. 37: 427-430, 1989.A.M. Rosenfalck, I. Bendtson, S. Jorgensen and C. Binder. Nasal glucagonin the treatment of hypoglycaemia in type 1 (insulin-dependent) diabeticpatients. Diabetes Research and Clinical Practice. 17: 43-50, 1992.

F.M. Sakr. Nasal administration of glucagon combined withdimethyl-β-cyclodextrin: comparison of pharmacokinetics andpharmacodynamics of spray and powder formulations. Int. J. Pharmaceutics132: 189-194, 1996.

Sibley et al. Prehosp. Emer. Care. Vol. 17: 98-102, 2013.

E. Stenninger and J. Aman. Intranasal glucagon treatment relieveshypoglycaemia in children with type 1 (insulin-dependent) diabetesmellitus. Diabetologia. 36: 931-935, 1993.

D. Teshima, A. Yamauchi, K.Makino, Y. Kataoka, Y. Arita, H. Nawata andR. Oishi. Nasal glucagon delivery using microcrystalline cellulose inhealthy volunteers. Int. J. Pharm. 233: 61-66, 2002.

D. P. Tieleman, D. van der Spoel and H.J.C. Berendsen. Moleculardynamics simulations of dodecyl phosphocholine micelles at threedifferent aggregate sizes: micellar structure and lipid chainrelaxation. J. Phys. Chem. B 104:6380-6388, 2000

O. Yanai, D. Pilpel, I. Harman, E. Elitzur-Leiberman and M. Philip. IDDMpatients’ opinions on the use of Glucagon Emergency Kit in severeepisodes of hypoglycaemia. Practical Diabetes Int. 14: 40-42, 1997.

SEQUENCES

(SEQ ID NO: 1)

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu- Met-Asn-Thr

We claim:
 1. A powder composition comprising glucagon (SEQ ID NO: 1),phospholipid surfactant and cyclodextrin, wherein at least a portion ofthe powder composition is present in a phase characterized by an XRPDmesopeak as determined by x-ray powder diffraction, and wherein saidpowder composition is prepared according to the method comprising thesteps of: a. forming a first solution of the glucagon (SEQ ID NO. 1) andthe phospholipid in an aqueous carrier, wherein the phospholipid ispresent in a concentration greater than or equal to the critical micelleconcentration; b. adding the cyclodextrin to the first mixture to form asecond mixture; c. drying the second mixture to form a solidformulation; and d. processing the solid formulation to produce auniform powder, said uniform powder including at least a portion of thepowder in a phase characterized by an XRPD mesopeak.
 2. The powdercomposition of claim 1 wherein powder composition comprises 5 to 15 wt%glucagon (SEQ ID NO: 1), 5 to 51 wt% of phospholipid surfactant and 44to 90 wt% of cyclodextrin.
 3. The powder composition according to claim2, wherein the phospholipid surfactant is selected from the groupconsisting of dodecylphosphocholine, didecylphosphatidylcholine,lysolauroylphosphatidylcholine, dioctanoylphosphatidylcholine anddilauroylphosphatidylglycerol.
 4. The powder composition according toclaim 2, wherein the cyclodextrin is an α-cyclodextrin, a β-cyclodextrinor a γ-cyclodextrin.
 5. The powder composition according to claim 4,comprising 90% to 70% by weight of cyclodextrin.
 6. The powdercomposition according to claim 4, comprising 90% to 70% by weight ofβ-cyclodextrin.
 7. The powder composition according to claim 2, furthercomprising up to 10 wt% of the overall weight of the composition ofsodium citrate or citric acid.
 8. The powder composition according toclaim 2, wherein the drying of the second mixture is carried out byfreeze drying or spray drying the second mixture.
 9. A nasal applicatorfor a powder composition, said applicator comprising a powderformulation reservoir, and further comprising the powder composition ofclaim 2 contained within the reservoir.
 10. A method for treatinghypoglycemia in an individual suffering from hypoglycemia comprisingadministering to the individual the powder composition of claim 2,wherein the composition is administered in a therapeutically effectiveamount as a powder to the nasal mucosa of the individual.
 11. A powdercomposition comprising glucagon (SEQ ID NO: 1), wherein said powdercomposition is prepared according to the method comprising the steps ofa. forming a first solution of the glucagon (SEQ ID NO. 1) and thephospholipid in an aqueous carrier, wherein the phospholipid is presentin a concentration greater than or equal to the critical micelleconcentration; b. adding the cyclodextrin to the first mixture to form asecond mixture; c. drying the second mixture to form a solidformulation; and d. processing the solid formulation to produce auniform powder.
 12. The powder composition of claim 11 wherein powdercomposition comprises 5 to 15 wt% glucagon (SEQ ID NO: 1), 5 to 51 wt%of phospholipid surfactant and 44 to 90 wt% of cyclodextrin.
 13. Thepowder composition according to claim 12, wherein the phospholipidsurfactant is selected from the group consisting ofdodecylphosphocholine, didecylphosphatidylcholine,lysolauroylphosphatidylcholine, dioctanoylphosphatidylcholine anddilauroylphosphatidylglycerol.
 14. The powder composition according toclaim 12, wherein the cyclodextrin is an α-cyclodextrin, aβ-cyclodextrin or a γ-cyclodextrin.
 15. The powder composition accordingto claim 14, comprising 90% to 70% by weight of cyclodextrin.
 16. Thepowder composition according to claim 14, comprising 90% to 70% byweight of β-cyclodextrin.
 17. The powder composition according to claim12, further comprising up to 10 wt% of the overall weight of thecomposition of sodium citrate or citric acid.
 18. The powder compositionaccording to claim 12, wherein the drying of the second mixture iscarried out by freeze drying or spray drying the second mixture.
 19. Anasal applicator for a powder composition, said applicator comprising apowder formulation reservoir, and further comprising the powdercomposition of claim 2 contained within the reservoir.
 20. A method fortreating hypoglycemia in an individual suffering from hypoglycemiacomprising administering to the individual the powder composition ofclaim 12, wherein the composition is administered in a therapeuticallyeffective amount as a powder to the nasal mucosa of the individual.